Structural studies of enoyl acyl carrier protein reductase from Plasmodium falciparum and Toxoplasma gondii.

Enoyl acyl carrier protein reductase enzyme (ENR) catalyses one of the two reductive steps in fatty acid elongation within the fatty acid synthesis type II cycle that is common to plants and prokaryotes. Since enzymes of this pathway are absent in humans they have become the target for several potent antibacterial compounds including triclosan which inhibits ENR in the picomolar range. As part of this thesis the gene for a type II ENR was located in the genomes of the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii. Analysis of the derived protein sequences suggested that these enzyme reside in the apicoplast. X-ray crystallographic techniques have been used to solve the structure for Plasmodium falciparum (Pf) and Toxoplasma gondii (Tg) ENR in complex with the NAD+ cofactor and triclosan by molecular replacement to 2.2A and 2.6A, respectively. Both enzymes. are tetrameric with the approximate dimensions of 90A x 90A x 50A. Each subunit is formed by a 7 stranded parallel β-sheet flanked by 9α helices, reminiscent of a Rossmann nucleotide binding fold common to several NAD+ binding enzymes. Analysis of the ENR family reveals that a characteristic of apicomplexan ENRs is an insert which varies in size from 42 residues in the P jalciparum enzyme to 6 residues in T.gondii ENR and which flanks the inhibitor/substrate binding site. In PfENR this loop is disordered but in the structure of TgENR the loop can be clearly seen and the structure shows that the loop lies close to the bound inhibitor but makes no direct contacts. Comparisons of the binding sites of a range of different ENR inhibitor complexes has led to a better understanding of the plasticity of the enzyme in response to inhibitor (and possibly substrate) binding. Moreover analysis of the substrate/inhibitor binding pocket in P jalciparum and T.gondii ENR shows that whilst they are similar to the bacterial enzymes there are distinct differences which could be exploited for the development of novel antiparasitic agents. A major hurdle in the delivery of inhibitors targeted towards the apicoplast organelle is the need to cross several barriers including the parasite membranes and host cell walls. However the addition of a releasable eight arginine linker to the phenolic OH group of triclosan significantly improved the speed of delivery and enabled triclosan to enter both the extracellular and intracellular T.gondii tachyzoites and bradyzoites. The identification of both a novel inhibitor for the apicomplexan family and a possible general delivery mechanism may provide a foundation for the development of ENR inhibitors that will efficiently treat several key parasitic diseases